title: MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.
abstract: Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled.
authors.affiliation: School of Biological and Biomedical Sciences, Durham University, South Road, Durham DH1 3LE, UK.
authors.forename: Hong Ting
authors.initials: HT
authors.lastname: Lin
authors.forename: Vassiliy N
authors.initials: VN
authors.lastname: Bavro
authors.forename: Nelson P
authors.initials: NP
authors.lastname: Barrera
authors.forename: Helen M
authors.initials: HM
authors.lastname: Frankish
authors.forename: Saroj
authors.initials: S
authors.lastname: Velamakanni
authors.forename: Hendrik W
authors.initials: HW
authors.lastname: van Veen
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.forename: M Inês
authors.initials: MI
authors.lastname: Borges-Walmsley
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Walmsley
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001703:Biophysics; D002462:Cell Membrane / Q000378:metabolism*; D004917:Erythromycin / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D018942:Macrolides / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D055503:Protein Multimerization
chemical_list: D018528:ATP-Binding Cassette Transporters; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C436312:MacA protein, E coli; C436313:MacB protein, E coli; D018942:Macrolides; D026901:Membrane Transport Proteins; C073126:tolC protein, E coli; D004917:Erythromycin; D000251:Adenosine Triphosphatases